TY - JOUR
T1 - The use of GAP-43 mRNA quantification in high throughput screening of putative neuroprotective agents in dorsal root ganglion cultures
AU - Keswani, Sanjay C.
AU - Rosenberg, Brian
AU - Hoke, Ahmet
N1 - Funding Information:
This work was supported by NIH (RO1 NS43991).
Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2004/7/30
Y1 - 2004/7/30
N2 - Large scale screening for neuroprotective drugs for peripheral neuropathies requires development of a high throughput system that is reliable and reproducible. Currently most accurate outcome measures of axonal degeneration are based on time-consuming, laborious measurement of morphological changes in neurites. In order to improve on the scalability of the screening procedure we developed a real-time RT-PCR based method of gene expression that correlates very well with morphological measures of neuritic degeneration. We examined the changes in GAP-43 expression in primary dorsal root ganglion (DRG) neurons in vitro with exposure to a zalcitabine (ddC), an antiretroviral drug that causes neuropathy in human immunodeficiency virus (HIV)-infected individuals, with and without FK506, an immunophilin ligand with neuroprotective and neuroregenerative properties. Similar to morphological measures of neuritic degeneration, in ddC-treated cultures there was a reduction in the expression of GAP-43 mRNA. This was prevented, in a dose-dependent manner, by co-administration of FK506. This assay, performed in a 96-well format, can easily be scaled for high throughput screening (HTS) using robotic systems.
AB - Large scale screening for neuroprotective drugs for peripheral neuropathies requires development of a high throughput system that is reliable and reproducible. Currently most accurate outcome measures of axonal degeneration are based on time-consuming, laborious measurement of morphological changes in neurites. In order to improve on the scalability of the screening procedure we developed a real-time RT-PCR based method of gene expression that correlates very well with morphological measures of neuritic degeneration. We examined the changes in GAP-43 expression in primary dorsal root ganglion (DRG) neurons in vitro with exposure to a zalcitabine (ddC), an antiretroviral drug that causes neuropathy in human immunodeficiency virus (HIV)-infected individuals, with and without FK506, an immunophilin ligand with neuroprotective and neuroregenerative properties. Similar to morphological measures of neuritic degeneration, in ddC-treated cultures there was a reduction in the expression of GAP-43 mRNA. This was prevented, in a dose-dependent manner, by co-administration of FK506. This assay, performed in a 96-well format, can easily be scaled for high throughput screening (HTS) using robotic systems.
KW - GAP-43
KW - High throughput assay
KW - Immunophilin ligand
KW - Neuroprotection
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U2 - 10.1016/j.jneumeth.2004.01.004
DO - 10.1016/j.jneumeth.2004.01.004
M3 - Article
C2 - 15183271
AN - SCOPUS:2942623919
SN - 0165-0270
VL - 136
SP - 193
EP - 195
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
IS - 2
ER -